Understanding the role of KLHL41 interactors in nemaline myopathy

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http://hdl.handle.net/2144/16822

Abstract

Nemaline Myopathy is a congenital disorder that is characterized by muscle weakness and limited mobility. Clinically, Nemaline Myopathy is highly heterogeneous, ranging from severe congenital forms with neonatal death to more mild childhood and adult onset forms. Mutations in 10 different genes have been linked to Nemaline Myopathy in human patients (ACTA1, NEB, TPM3, TPM2, CFL2, TNNT1, LMOD3, KBTBD13, KLHL40, and KLHL41). Approximately 75% of Nemaline Myopathy cases with a known genetic diagnosis are caused by mutations in structural proteins within the thin filaments in skeletal muscle. It is very difficult to develop therapies that target structural proteins and there are no effective, specific therapies for Nemaline Myopathy. Recent discovery of mutations in Kelch family genes, which are non-structural genes, provide an alternative pathway for developing targeted treatments for Nemaline Myopathy.
Of particular interest in this study is the protein KLHL41. Mutation of KLHL41 has shown a clear genotype-phenotype relationship in Nemaline Myopathy with frameshift mutations resulting in severe forms and missense mutations resulting in typical congenital forms. KLHL41 is found primarily in striated muscle and plays a role in protein turnover through the ubiquitin-proteasome pathway. KLHL41 interacts with Cullin 3 and serves as a substrate adapter in the E3 ligase complex, the last step in ubiquitination of proteins targeted for degradation by proteasomes. Preliminary studies demonstrate that KLHL41 interacts with nebulin, a major structural protein in muscle. Mutations in the nebulin gene are implicated in 50% of Nemaline Myopathy cases. KLHL41 has also been shown to interact with POMP, a protein that facilitates the formation of the 20S proteasome subunit. The proteasome pathway has been shown to be impaired in Nemaline Myopathy patients, regardless of genetic mutation. A better understanding of how the proteasome pathway becomes disrupted in Nemaline Myopathy will contribute to the development of specific targeted therapies.
This study sought to understand the significance of KLHL41-protein interactions on disease pathology in Nemaline Myopathy. Co-transfection of increasing amounts of KLHL41 with constant levels of POMP or a nebulin fragment showed a decrease in POMP and nebulin expression, suggesting that KLHL41 is a negative regulator of POMP and nebulin. Co-transfection and immunoprecipitation were also performed to confirm the direct interaction between KLHL41 and POMP or nebulin. However, this study could not re-confirm a direct physical interaction between KLHL41 and POMP or nebulin identified by previous Y2H and HTRF studies.
As a substrate adapter in the E3 ligase complex, KLHL41 plays an important role in protein turnover and disruption of this pathway may contribute to the pathology of Nemaline Myopathy. Confirming the role of KLHL41 in the development of Nemaline Myopathy is critical to development of future therapeutic strategies.